Project Summary Around 90,000 cases of melanoma, the deadliest form of skin cancer, will be diagnosed in the United States in the coming year. For very early stage melanoma diagnosis, 5-year survival is around 95%. This number drops to 62% for melanoma that has spread to nearby lymph nodes, and for late stage melanoma the 5-year survival rate is only 15-20%. Recently, immunotherapy drugs targeting immune cell inhibitory receptors on T cells have been successfully developed for the treatment of melanoma and a number of other cancers. Many of these therapies target programmed death-1 (PD-1), an inhibitory receptor that decreases T cell activation. While most of these therapies focus on T cells, comparably less research has been done on using natural killer (NK) cells to better combat cancer. NK cells are intriguing targets for immunotherapy for a number of different reasons, including their cytotoxic abilities and relatively short lifespan. In addition, NK cells have been shown in vivo to contribute to cancer surveillance. Although great strides have been made in immunotherapy research, many forms of cancer escape current immunotherapy drugs. Thus, novel therapeutic targets need to be identified for the development of new treatments. One such target is killer cell lectin-like receptor G1 (KLRG1), which is a well conserved inhibitory molecule expressed on NK cells. KLRG1 has two main ligands, E-cadherin and N-cadherin. Engagement of KLRG1 inhibits IFN-? and TNF-? production and high KLRG1 expression has been correlated with low proliferative capacity. To determine the role of KLRG1 as a potential NK cell checkpoint, we developed mice globally deficient for KLRG1 and mice conditionally deficient for KLRG1 in the NK cell lineage and took advantage of the NK sensitive cancer model, B16. Following B16-E-cadherin administration, we found that KLRG1 deficient animals had!significantly fewer tumors in the lung tissue, compared to wild type controls. In addition, compared to the parental B16 cell line, B16-E-cadherin resulted in a higher number of tumors in wild- type mice. Based on this data, I hypothesize that targeting KLRG1 will unleash the immune cell response to cancer. In Specific Aim 1, I will determine if targeting KLRG1 on NK cells will reduce tumor development and progression. In Specific Aim 2, I will determine if simultaneous KLRG1 and PD-1 blockade can synergize to increase immune cell anti-cancer activity. The findings generated from the proposed work could potentially lead to the development of new checkpoint inhibitors to treat cancer. !